The relationship between concentration of a dual marker strain of Salmonella Typhimurium in bovine faeces and its probability of detection by immunomagnetic separation and culture

AIMS: To modify a strain of Salmonella serotype Typhimurium to express unique marker traits and then define how the concentration of the marker in bovine faeces affects the probability of its detection by culture preceded by immunomagnetic separation (IMS). METHODS AND RESULTS: DNA encoding for the production of green fluorescent protein (gfp) and resistance to kanamycin was inserted into the bacterial chromosome of Salm. Typhimurium. Transposon insertion was demonstrated by Southern blot hybridization. Varying amounts of one electroporant (gfpSal-1) were inoculated into suspensions of bovine faeces and attempts made to isolate gfpSal-1 using a protocol based on pre-enrichment incubation, IMS and enrichment in selective media. Isolates of gfpSal-1 were differentiated from wild strains of Salmonella using fluorescence under u.v. light and expression of kanamycin resistance. A logistic and Gompertz function each derived from the dose-response data partially explained the observations with the fit of the Gompertz function judged to be superior. The 10, 50 and 90% limits of detection from the Gompertz function were estimated to be 1.92, 2.03 and 2.27 CFU g(-1) respectively. CONCLUSIONS: Reliance on the traditional concept of 'limit of detection' could introduce unacceptable errors in the interpretation of test findings when the concentration of Salm. Typhimurium in bovine faeces (pooled or individual) is below ca 3 CFU g(-1) of faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The dose-response curve can be used to aid the design of protocols for detecting Salmonella in individual and pooled faecal specimens. The experiments demonstrate that both reporter genes in tandem are useful for studying the performance of culture-based methods for detecting pathogens in faeces.     

Evaluation of an indirect serum ELISA and a bacteriological faecal culture test for diagnosis of Salmonella serotype Dublin in cattle using latent class models

AIMS: To evaluate a conventional bacteriological test based on faecal culture and an indirect serum ELISA for detection of S. Dublin infected cattle. To compare the predictive values of the two tests in relation to the prevalence. METHODS AND RESULTS: A total of 4531 paired samples from cattle in 29 dairy herds were analysed for presence of S. Dublin bacteria in faeces and immunoglobulins directed against S. Dublin lipopolysaccharide in an indirect serum ELISA. Sensitivity and specificity were estimated at two ELISA cut-off values using a validation method based on latent class models, which presumably provides less biased results than traditional validation methods. Stratification of data into three age groups gave significantly better estimates of test performance of the ELISA. Receiver operating characteristic (ROC) curves were constructed for comparison of overall performance of the ELISA between the three age groups. The sensitivity of the faecal culture test was low (6-14%). ELISA appeared to have a higher validity for animals aged 100-299 days of age than older or younger animals. Overall, the negative predictive value of the ELISA was 2-10 times higher than for the faecal culture test at realistic prevalence of infection in the test population. CONCLUSIONS: The diagnostic sensitivity of the faecal culture test for detection of S. Dublin is poor, the specificity is 1. The superior sensitivity and negative predictive value of the serum ELISA makes this test preferable to faecal culture as an initial screening test and for certification of herds not infected with S. Dublin. SIGNIFICANCE AND IMPACT OF THE STUDY: A quantitative estimate of the sensitivity of a faecal culture test for S. Dublin in a general population was provided. ELISA was shown to be an appropriate alternative diagnostic test. Preferably, samples from animals aged 100-299 days of age should be used as these give the best overall performance of the ELISA. Plots of ROC curves and predictive values in relation to prevalence facilitates optimisation of the ELISA cut-off value.     

Use of IgG avidity ELISA to differentiate acute from persistent infection with Salmonella Dublin in cattle

AIMS: To investigate whether an immunoglobulin (Ig)G avidity ELISA can be used to differentiate between acute and persistent infection with Salmonella (S.) Dublin in cattle. To determine whether the IgG isotype, IgG(1) and IgG(2) responses in acute and persistent infections differ. METHODS AND RESULTS: Animals were selected from two herds with long-term infection (years) and two herds recently infected (<3 months). Forty-seven animals were categorized into groups based on the persistence of their antibody level in milk. Based on titre from two serial dilutions the avidity index (AI) was calculated for IgG (IgG-AI), IgG(1) (IgG(1)-AI) and IgG(2) (IgG(2)-AI). The mean IgG-AI for suspected carrier animals with either persistently high (group 1) or persistently high to medium high (group 2) antibody levels was significantly (P = 0.003) higher (32.1% and 38.4%) than for acutely infected animals (21.7% and 22.3%). The probability of being a suspect carrier was associated with IgG-AI, antibody level in the sample and age. However, the effect of age could be the result of a biased sample selection. Specificities and sensitivities were calculated at a range of cut-off values for IgG-AI and IgG(1)-AI. Overall, IgG(2)-AI was high compared with IgG(1)-AI, and there was no difference in IgG(2)-AI between infection groups. There was no difference in the ratio IgG(2):IgG(1) for acute and persistent infection groups. CONCLUSIONS: Assuming that a persistently high antibody response is indicative of persistent infection with S. Dublin in cattle, it can be concluded that the IgG-AI can aid in differentiating between acute and long-term infection on herd level. However, for the test to be useful as an alternative tool to repeated sampling over time for detection of persistently infected carriers during control strategies in cattle herds, the test needs to be optimized and studied further in a larger sample of well-characterized infections in cattle. The affinity of IgG(2) is higher than IgG(1) early in the S. Dublin infection. There appears to be no difference in the IgG(2)-AI between the acute and chronic infection stages. SIGNIFICANCE AND IMPACT OF THE STUDY: For decades the strategies for detection of persistently infected cattle in S. Dublin infected herds have involved repeated bacteriological culture of faecal samples or repeated antibody measurements over several months. Both methods are time consuming and costly, leaving a new method for detection of carrier animals based on a single sampling highly desirable. This study illustrates a tool, IgG-AI, which may prove useful, although more validation of the method is required before it is used in practice.     

不同增菌和培养条件下沙门氏菌检出效果的比较

通过在鸡精样本中添加低含量的鼠伤寒沙门氏菌和干扰菌(弗氏柠檬酸杆菌和奇异变形杆菌),参考国标和美国药典中沙门氏菌的定性方法,研究比较了3种选择性增菌液、4个培养时间段以及4种选择性平板对于沙门氏菌的不同检出效果。结果表明,在RV肉汤及四硫磺酸钠煌绿增菌液(TTB)中培养18 h～20 h,使用沙门显色培养基及亚硫酸铋琼脂(BS平板)进行选择性培养沙门氏菌的检出效果最好。     

A comparative evaluation of the sensitivity of Salmonella detection on processed chicken carcasses using Australian and US methodologies

Aim: To confirm the reliability and sensitivity of Salmonella testing of processed poultry in Australia. Methods and Results: The detection of Salmonella in a whole carcass wash of 90 randomly selected processed broilers was compared using the Australian Standard method, an Australian industry method used by a major processor and the United States Department of Agriculture method published in the Federal Register. The sensitivity of each method was determined using a carcass wash containing a known number of Salmonella Typhimurium to determine the minimum concentration to be able to be identified as positive. The two Australian methods were found to be comparable with both the Australian methods detecting more positive carcasses than the United States Department of Agriculture (USDA) method. The Australian methods were sensitive at the level of 1–3 CFU ml^?1 and the USDA method was sensitive at 10–30 CFU ml^?1. Conclusions: The Australian Standard method and the Australian industry method were both able to detect Salmonella reliably even at a low level of contamination. Significance and Impact of the Study: This study gives a high level of confidence both to the operators of poultry‐processing plants and to regulators dependent upon the outcome of Salmonella testing for process control in Australia.     

Application of BAX system, Tecra Unique Salmonella test, and a conventional culture method for the detection of Salmonella in ready-to-eat and raw foods

AIMS: To compare the BAX system, the Tecra Unique Salmonella test, and a conventional culture method for the detection of Salmonella in various foods. METHODS AND RESULTS: Ready-to-eat and raw foods were inoculated with Salmonella serotype Typhimurium, Salmonella serotype Enteritidis, Salmonella serotype Typhi, or Salmonella serotype Derby. Incubated pre-enrichment cultures were examined using the BAX system, the Tecra Unique Salmonella test, and a conventional culture method. Salmonella could be detected in all ready-to-eat food samples inoculated with S. Typhimurium, S. Enteritidis, or S. Derby, with any of the three test methods. However, false negatives were obtained with the Tecra test and the culture method when samples with higher background flora were inoculated with S. Typhi. Sensitivity test results suggested the two rapid tests performed as well as the culture method in the detection of 10(1) CFU of S. Typhimurium in 25-g cooked or raw food. CONCLUSIONS: The BAX system and the Tecra Unique Salmonella test demonstrated results comparable with those of the culture method in the detection of Salmonella serotypes used except S. Typhi. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first evaluation of the BAX system, the Tecra Unique Salmonella test, and a culture method in the detection of Salmonella in a variety of western and oriental foods.     

A novel multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces

AIMS: To develop a multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces. METHODS AND RESULTS: A total of 54 Salmonella strains representing 19 serovars and non-Salmonella strains representing 11 different genera were used. Five primer pairs were employed in the assay. Three of them targeted to the genes hilA, spvA and invA that encode virulence-associated factors. A fourth primer pair amplified a fragment of a unique sequence within S. enterica serovar Enteritidis genomes. An internal amplification control (a fragment of a conservative sequence within the 16S rRNA genes) was targeted by a fifth primer pair. The assay produced two or three amplicons from the invA, hilA and 16S rRNA genes for 19 Salmonella serovars. All Salmonella and non-Salmonella strains yielded a band of an internal amplification control. For S. enterica serovar Typhimurium, four products (the fourth from the spvA gene), and for S. enterica serovar Enteritidis five amplicons (the fifth from the sdf gene) were observed. S. enterica serovar Enteritidis was cultured from three of 71 rectal swabs from diarrhoeal patients. Five specific amplicons were generated with the multiplex PCR assay only from culture-positive faecal samples. CONCLUSION: The multiplex PCR assay specifically detects S. enterica serovar Enteritidis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a novel multiplex PCR assay, which contains an internal amplification control and enables concurrent survey for Salmonella virulence genes.     

The prevalence of Salmonella spp. in bovine faecal,rumen and carcass samples at a commercial abattoir

AIMS: To determine the prevalence, serotype and antibiotic resistance profile of Salmonella isolates in cattle and on carcasses at a commercial Irish abattoir. METHODS AND RESULTS: Faecal, rumen and carcass samples were collected from a beef abattoir over a 12-month period and examined for the presence of Salmonella spp. Isolates were serotyped, phage typed (when serotype was found to be S. Typhimurium) and tested for susceptibility to a panel of antibiotics. Salmonella was isolated from 2% of faecal, 2% of rumen and 7.6% of carcass samples. Salmonella was most frequently isolated from samples taken during the period August to October. S. Dublin was isolated from 72% of positive samples. S. Agona and S. Typhimurium definitive type (DT)104 were each isolated from 14% of positive samples. All S. Typhimurium DT104 isolates were resistant to ampicillin, chloramphenicol, streptomycin, sulphafurazole and tetracycline (ACSSuT). On occasion, from a single animal, the same serotype was isolated from more than one sample (i.e. faeces and rumen; faeces and carcass; rumen and carcass; faeces, rumen and carcass). CONCLUSIONS: Salmonella is present in cattle at slaughter and on beef carcasses at an Irish abattoir, with a higher frequency of occurrence during the period August to October. Most isolates from the study are not commonly associated with human clinical infection, with the exception of S. Typhimurium DT104 (R-type ACSSuT). SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides epidemiological data that is necessary for the understanding of beef as a source of human Salmonella infection.     

Eight-hour PCR-based procedure for the detection of Salmonella in raw oysters

The aim of this study was to evaluate the ability of a nested PCR system to detect Salmonella senftenberg in raw oysters. The specific primers of the PCR were derived from the invA gene sequence, essential for Salmonella invasiveness into epithelial cells. First, for the extraction of DNA, four methods (guanidine isothiocyanate, E.Z.N.A. Mollusc Kit, Chelex-100, and lysis with detergents) were compared. A nested PCR method combined with 3.5 h pre-enrichment in buffered peptone water (BPW) and DNA extraction by the resin Chelex-100 is proposed for the detection of S. senftenberg in oyster samples. The detection limit of the method is less than 0.1 CFU/ml (<1 CFU/g of oyster). This procedure is shown to be an excellent tool for the sensitive detection of S. senftenberg from naturally contaminated oysters, with results being obtained within 8 h.     

Transmission of Salmonella between wildlife and meat-production animals in Denmark

Aims: To investigate the transmission of Salmonella spp. between production animals (pigs and cattle) and wildlife on production animal farms in Denmark. Methods and Results: In the winter and summer of 2001 and 2002, 3622 samples were collected from Salmonella-infected and noninfected herds of pigs and cattle and surrounding wildlife. Salmonella was detected in wildlife on farms carrying Salmonella-positive production animals and only during the periods when Salmonella was detected in the production animals. The presence of Salmonella Typhimurium in wild birds significantly correlated to their migration pattern and food preference. Conclusions: Salmonella was transmitted from infected herds of production animals (cattle and pigs) to wildlife that lived amongst or in close proximity to them. Significance and Impact of the Study: Salmonella in animal food products is associated with the occurrence of Salmonella in primary animal production. Strategies to control the introduction and spread of infection should include wildlife management, as the nearby wildlife may act as reservoirs for Salmonella spp. and/or may be passive carriers of the bacteria.     

Immunomagnetic separation and conventional culture procedure for detection of naturally occurring Salmonella in raw pork sausages and chicken meat

The aim of the study was to compare immunomagnetic separation (IMS) and conventional selective enrichment procedures using selenite cystine broth (SC) and Rappaport–Vassiliadis broth (RV) in 137 naturally contaminated food samples (69 raw pork sausages and 68 chicken meat). The utilization of SC or IMS appeared to be the most appropriate enrichment procedure: 15 out of 18 Salmonella -positive samples (83·3%) were detected by SC and 12 (66·7%) by IMS; RV yielded only seven positive isolations (38·9%). However, RV yielded the highest count of Salmonella colonies per plate and the lowest interference by competing organisms. IMS could become a reliable alternative to standard enrichment procedures and a combined IMS and selective enrichment broth could increase the chance of Salmonella recovery.     

Evaluation of culture, ELISA and PCR assays for the detection of Salmonella in seafood

Aims: The study evaluated the efficiency of culture, enzyme‐linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) assays for the detection of Salmonella in naturally contaminated seafood. Methods and Results: In this study, 215 seafood samples comprising fish, shrimp, crab, clam, mussel, oyster, squid, cuttlefish and octopus from fish market of Cochin (India), were compared by culture, ELISA and PCR methods. Bacteriological Analytical Manual (BAM), U.S. Food and Drug Administration (USFDA) method was followed for culture assay, and Salmonella Tek, a commercial sandwich ELISA kit, was used for ELISA assay. Salmonella‐specific PCR assay was developed for 284 bp Salmonella‐specific invA gene amplicon. PCR assay exhibited 31·6% seafood positive for Salmonella followed by ELISA (23·7%) and culture method (21·3%). There was fair to excellent agreement between culture, ELISA and PCR assays (kappa coefficient values ranging from 0·385 to 1·0) for different seafood samples. Conclusion: The investigation revealed the greater concordance between culture and ELISA methods for seafood. Among the three methods, PCR assay was most sensitive. Lower detection rate with culture and ELISA assays could be attributed to greater sensitivity of the PCR method in the detection of Salmonella in seafood. Significance and Impact of the Study: We propose the incorporation of dual tests based on different principle and procedure for the routine analysis of Salmonella in seafood.     

重组酶介导等温核酸扩增方法快速检测土壤沙门氏菌

【背景】沙门氏菌(Salmonella)是一种可以引起人畜患病的致病菌,也是最主要的食源性细菌之一。土壤中的沙门氏菌可通过蔬菜等植物进入人体,引发食物中毒。但由于土壤性质及其他微生物的干扰,如何快速甄别土壤是否受到沙门氏菌的污染仍是一个难题。【目的】建立一种快速、灵敏检测土壤沙门氏菌的实时重组酶介导等温核酸扩增(Real-Time Recombinase Aided Amplification,RT-RAA)方法。【方法】针对沙门氏菌invA基因序列设计特异性引物和探针,构建含有invA基因待检片段的重组质粒,评价RT-RAA方法的灵敏度;分别以肠炎沙门氏菌、大肠杆菌、福氏志贺氏菌和金黄色葡萄球菌的基因组DNA为模板,评价RT-RAA方法的特异性;RT-RAA方法用于番茄、生姜土壤中沙门氏菌的检测,同时用平板培养法进行验证。【结果】RT-RAA方法可用于重组质粒中invA基因片段的检测,在39℃条件下,20 min内即可获得检测结果,最低检测质粒拷贝数为10拷贝/反应,而且与大肠杆菌、福氏志贺氏菌和金黄色葡萄球菌无交叉反应。土壤样品DNA的RT-RAA检测结果显示,供试番茄土已被沙门氏菌污染,而生姜土则没有,与平板培养结果一致。【结论】RT-RAA方法具有灵敏度高和特异性强的特点,可用于土壤沙门氏菌污染的快速检测。     

Growth factor requirements of organogenesis in serum-free metanephric organ culture

Summary In order to define humoral growth factors which may regulate mammalian renal development, the growth requirements of fetal metanephric organogenesis were studied in serum-free murine organ culture. Metanephric growth, determined by cell proliferation and protein content, and metanephric differentiation, determined morphometrically as epithelial glomerular formation, were compared and contrasted following 144 hours of organ culture incubation in basal medium, basal medium supplemented with 10% fetal bovine serum, and basal medium supplemented with various combinations of growth factors. The basal medium was composed of equal volumes of Dulbecco's modified Eagle's medium and Hams' F-12 medium. Five humoral growth factors were studied in the following concentrations: selenium, 6.8×10⁻⁹ M; insulin, 8.3×10⁻⁷ M; triiodothyronine, 2×10⁻⁹ M; transferrin, 6.2×10⁻⁸ M; and prostaglandin E₁, 7.1×10⁻⁸ M. Results showed that transferrin and prostaglandin E₁ were necessary for optimal growth in the system and that prostaglandin E₁ was necessary for maximal metanephric differentiation. Such data provide guidelines for the creation of serum-free medium for future fetal renal cell and tissue culture systems, and provide insight into the factors which may regulate normal and abnormal renal embryogenesis and the reparative processes of renal hyperplasia and hypertrophy which follow renal injury. A preliminary report of this work was presented at the Ninth International Congress of Nephrology, Los Angeles, June 1984. These studies were supported in part by Basil O'Connor Starter Research Grant 5-349 from the March of Dimes Birth Defects Foundation and New Investigator Research Award I-R23-AM34891-01 from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases of the National Institutes of Health (Both to Dr. Avner). Editor's Statement The determination of effects of extracellular components on the introduction and maintenance of differentiation is an area for which serum-free culture techniques are particularly suited. The approaches described in this report utilize morphometric techniques to quantitate differentiation in serum-free fetal organ culture in addition to standard methodologies for assessing growth. The purely epithelial nature of the cultures used in these studies also provides some interesting advantages in the design of experiments aimed, at determining the importance of cell-cell interactions at various stages in the differentiative process. David W. Barnes     

需氧厌氧培养方式对甲型副伤寒沙门菌检出率的影响

目的探讨需氧和厌氧血培养方式的选择对甲型副伤寒沙门菌检出率的影响。方法使用mini VI-TAL自动荧光血培养仪或BacT/Alert 3D培养仪对18684例疑似血流感染患者血液(含骨髓)标本进行需氧和(或)厌氧培养(其中仅做需氧培养935例,仅做厌氧培养16例,需氧和厌氧配对培养17733例,每瓶注入约5 ml血液)。结果18684例血液标本,共分离到甲型副伤寒沙门菌3888例(20.81%)。在17733例需氧和厌氧配对培养中检出3613例,其中仅需氧阳性406例占11.24%(406/3613),仅厌氧阳性405例占11.21%(405/3613),需氧和厌氧培养均阳性者2802例占77.55%(2802/3613),需氧和厌氧均生长的阳性报警时间分别为(22.56±13.22)h和(26.69±15.80)h,仅需氧阳性的报警时间为(32.85±23.33)h,仅厌氧阳性的报警时间为(34.46±18.44)h。结论需氧和厌氧血培养方式获得甲型副伤寒沙门菌的阳性率相同,采用需氧和厌氧瓶配对培养可提高阳性检出率,只做厌氧或需氧培养将有11.24%或11.21%阳性病例被漏检。     

Growth and antrum formation of bovine primary follicles in long-term culture in vitro

Successful antral formation in vitro from bovine preantral follicles (145–170 μm) has been described previously, but antrum formation from the primary follicle (50–70 μm) has not yet been achieved in vitro. The aim of the study was to establish an optimal culture system supporting the growth and maturation of bovine primary follicles (50–70 μm) in vitro. Bovine primary follicles were cultured in a three-dimensional culture system for 13 or 21 days in alpha-minimum essential medium. Various treatments including follicle stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E₂), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were tested. The follicular diameter and antrum formation rate were recorded, and follicular maturation markers (P450 aromatase, CYP19A1; anti-Mullerian hormone, AMH; growth differentiation factor-9, GDF9; bone morphogenetic protein-15, BMP15; and type III transforming growth factor β receptor, TGFβR3) were analyzed by real-time RT-PCR. After 21 days of culture under each treatment condition, the follicular diameter was significantly enlarged in the presence of FSH + LH + E₂ + bFGF or FSH + LH + E₂ + bFGF + EGF (p < 0.05). An addition of 50 ng/ml bFGF or bFGF + 25 ng/ml EGF initiated antrum formation by day 19 and day 17 of culture, and the antral cavity formation rate was 16.7% and 33.3% by 21 days of culture, respectively. The expression of follicular maturation markers (CYP19A1, AMH, GDF9, BMP15 and TGFβR3) was significantly altered. We conclude that addition of 50 ng/ml bFGF + 25 ng/ml EGF to media containing FSH + LH + E₂ turned out to be the most effective optimized culture conditions to support the growth and maturation of bovine primary follicles in vitro.     

The synthesis of subendothelial matrix by bovine aortic endothelial cells in culture

Bovine aortic endothelial cells cultured on collagenous or plastic substrata continuously synthesize and deposit a subendothelial matrix, independently of whether the cells are in the logarithmic or the stationary phase of growth. This subendothelial matrix contains fibrillar and amorphous elements comparable with those observed in the subendothelium in vivo. Deposition of subendothelial matrix on a collagen gel substratum both started earlier and progressed at approximately double the rate than that on denatured collagen. The relative composition of the subendothelial matrix was assessed by sequential incubation with trypsin, elastase and collagenase (Jones et al., 1979). The subendothelial matrix deposited on collagen gels by early confluent cultures and late post-confluent cultures differed in their enzyme sensitivity. These age-related changes in the enzyme sensitivity of the subendothelial matrix were characteristic for each cloned cell population examined. Comparable variations in the composition of the subendothelial matrix were not observed when the cells were cultured on plastic or gelatin-coated dishes; the subendothelial matrix deposited on these two substrata contained considerably more trypsin-sensitive material and less elastase and collagenase-sensitive material than the matrix deposited on native collagen gels. Age-related changes in the enzyme sensitivity of the subendothelial matrix deposited on collagen gels was found to be a function of the time elapsed since confluence and it was not related to the time elapsed since plating or to the number of cells present.     

生物学因子对紫苏悬浮培养细胞生长和花色素形成的影响

应用摇瓶培养研究了生物学因子,即：细胞聚集体大小、继代周期和接种量,对紫苏悬浮细胞生长和次生代谢物花色素产生的影响。结果表明：与未经筛选的或细胞聚集体小于250μm的细胞团块相比,大于250μm的细胞团块作为接种细胞时,培养所得的花色素含量较低。7一10天为合适的继代周期,在长时期的继代过程中,细胞生长良好、并且色素含量也高。实验还表明,每升接种50克湿细胞最适合于细胞增殖与色素积累。